To study proteins and enzymes in their natural context in living organisms, a noninvasive imaging technique with high spatial and temporal resolution is required. Such resolution can be achieved using magnetic resonance imaging (MRI), which has been used extensively in the last two decades for anatomical, functional, and dynamic imaging. The imaging probes for magnetic resonance imaging (MRI) are termed “contrast agents,” since they enhance the water proton-based contrast between the imaging target and the surrounding tissue. Detection with MRI relies on contrast in the MRI signal between the tissue of interest and its surrounding tissue, which can be further enhanced by expression of certain exogenous proteins that increase MRI contrast.
Recently, a new type of MRI contrast that relies on direct chemical exchange of protons with bulk water has been developed, and is referred to as chemical exchange saturation transfer (CEST) MRI. CEST MRI is a technique in which low-concentration marker molecules are labeled by either saturating or labeling their exchangeable protons spins by radio-frequency (RF) irradiation. If such saturation or labeling can be achieved rapidly (i.e., before the spin exchanges), exchange of such labeled spins with water leads to transfer of the magnetization, allowing indirect detection of the solute via the water resonance through a change in signal intensity in MRI.
A variety of organic molecules possessing protons that exchange rapidly with the surrounding water protons have been suggested as new contrast agents. These exchangeable protons can be “magnetically tagged” using a radiofrequency saturation pulse applied at their resonance frequency. The tagged protons exchange with the protons of surrounding water molecules and consequently reduce the MRI signal. This in and of itself would not be visible at the low concentrations of solute, but the exchanged protons are replaced with fresh, unsaturated protons and the same saturation process is repeated. Over time (e.g., several seconds) this repetition results in signal amplification, and very low concentrations of agents can be detected. Hence, these agents are termed CEST contrast agents.
Each CEST contrast agent can have a different saturation frequency, which depends on the chemical shift of the exchangeable spin. The magnitude of proton transfer enhancement (PTE) due to this effect, and the resulting signal reduction from equilibrium (S0) to saturated (Ssat), are given by:
                                          P            ⁢                                                  ⁢            T            ⁢                                                  ⁢            E                    =                                                                      NM                  w                                ⁢                α                ⁢                                                                  ⁢                                  k                  ex                                                                                                  (                                          1                      -                                              x                        CA                                                              )                                    ⁢                                      R                                          1                      ⁢                      wat                                                                      +                                                      x                    CA                                    ⁢                                      k                    ex                                                                        ·                          {                              1                -                                  e                                                            -                                              [                                                                                                            (                                                              1                                -                                                                  x                                  CA                                                                                            )                                                        ⁢                                                          R                                                              1                                ⁢                                wat                                                                                                              +                                                                                    x                              CA                                                        ⁢                                                          k                                                              ex                                ⁢                                                                                                                                                                                                                                      ]                                                              ⁢                                          t                      sat                                                                                  }                                      ,                            [                  Eq          .                                          ⁢          1                ]                                                          ⁢                              and            ⁢                                                  ⁢                          (                              1                -                                                      S                    sat                                    /                                      S                    0                                                              )                                =                                                    P                ⁢                                                                  ⁢                T                ⁢                                                                  ⁢                                  E                  ·                                      [                    CA                    ]                                                                              2                ·                                  [                                                            H                      2                                        ⁢                    O                                    ]                                                      .                                              [                  Eq          .                                          ⁢          2                ]            “CA” is the contrast agent containing multiple exchangeable protons, xCA its fractional exchangeable proton concentration, α the saturation efficiency, k the pseudo first-order rate constant, N the number of exchangeable protons per molecular weight unit, and Mw the molecular weight of the CA. The exponential term describes the effect of back exchange and water longitudinal relaxation (Rlwat=1/Tlwat) on the transfer during the RF saturation period (tsat). This effect will be bigger when spins exchange faster, but the catch is that saturation must occur faster as well, which increases the radio-frequency power needed. In addition, the resonance of the particular spins must be well separated from the bulk in the NMR spectrum, which requires a slow exchange on the NMR time scale. This condition means that the frequency difference of the exchangeable spins with the bulk is much larger than the exchange rate (Δω>k).
Thus, the CEST technology becomes more applicable at higher magnetic fields or when using paramagnetic shift agents. Any molecule that exhibits a significant PTE effect can be classified as a CEST (contrast) agent. The concept of these agents as MR contrast agents is somewhat similar to the chemical amplification of colorimetric labels in in situ gene expression assays. For instance, CEST agents can be detected by monitoring the water intensity as a function of the saturation frequency, leading to a so-called Z-spectrum. In such spectra, the saturation effect of the contrast agent on the water resonance is displayed as a function of irradiation frequency.
Since the first report of CEST contrast in 2000, CEST MR imaging has become a widely used MRI contrast mechanism, and CEST contrast is generated by the dynamic exchange process between an exchangeable proton of a biomarker of interest and the surrounding water protons. To detect the biomarkers, the magnetization of some of their exchangeable protons is nullified by applying a selective radiofrequency saturation pulse at the specific resonance frequency (chemical shift) of the target protons. Due to exchange of the “saturated” agent protons with surrounding water protons, the net water signal is reduced, thus enhancing the MRI contrast.
Molecular MRI is an attractive technique for many applications because it can track metabolite dynamics in vivo with better spatial resolution and anatomical co-registration than traditional techniques. While CEST MRI has been employed for many applications in molecular and cellular MRI, there remains an urgent need for the design and development of MRI contrast agents that offer improved sensitivity and contrast effects in producing MR images.